GeXP Detection Kits for Identification of 11 Kinds of Duck Virus Diseases

a technology of duck virus and detection kits, applied in the field of biotechnology, can solve the problems of limiting the development of duck breeding, increasing the incidence of duck communicable virus diseases, and complicated operation of duck breeding

Active Publication Date: 2016-03-03
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032]The experiments in the present invention prove that it has a high specificity to detect simultaneously avian influenza virus, subtypes H5, H7 and H9 of avian influenza virus, duck hepatitis virus, duck enteritis virus, duck Tembusu virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus with the 12 prime pairs of reagent or detection kit based on GeXP system provided in the present invention, and the sensitivity thereof is respectively 10 copies / μL, 100 copies / μL, 100 copies / μL, 100 copies / μL, 10 copies / μL, 100 copies / μL, 10 copies / μL, 10 copies / μL, 100 copies / μL, 10 copies / μL, 100 copies / μL, 100 copies / μL, and compared with identification results with conventional experimental methods, such as virus isolation and hemagglutination inhibition test etc., the coincidence rate reaches 100%. The invention provides a simple, high-throughput detection kit and detection system for common and major duck communicable diseases and pathogens thereof, meeting with actual needs better and having a broad prospect of application.

Problems solved by technology

With the development of duck breeding, the incidence of duck communicable virus diseases is also gradually increasing, which has become an important factor which restricts the development of duck breeding.
Traditional methods of differential diagnosing these duck communicable diseases mainly include isolation and identification of pathogen, serological tests etc., while these methods are often limited by freshness of clinical samples, pollution degree of clinical samples, or course of disease of clinical samples, the operations thereof are also very complicated, time-consuming, furthermore, it is more difficult for these methods to differential diagnose multiplex infections.
PCR products need to be observed by agarose gel electrophoresis which is difficult to distinguish bands within 50 bp-100 bp, and generally only duplex-sextuple PCRs can be carried out, so it is difficult to achieve high throughput detection.
Multiplex fluorescent PCR generally can only detect quadruplex PCR, because probes need to be labeled with fluorescent groups with different light emitting wavelengths, if probes labeled with too many fluorescent groups, interference will emerge between each other, so multiplex fluorescent PCR can only detect duplex-quadruplex PCR.
Because several primer pairs are present in the multiplex PCR reaction system at the same time, the possibility of forming complex primer dimers greatly increases, and target gene number which can be detected simultaneously is limited (usually 2-6 genes), all these reasons cause that the multiplex PCR cannot achieve the goal of high throughput and rapid detection and analysis.
At present, there is no reagent or detection kit which can simultaneously detect multiple pathogens of communicable diseases from duck source based on GeXP system.

Method used

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  • GeXP Detection Kits for Identification of 11 Kinds of Duck Virus Diseases
  • GeXP Detection Kits for Identification of 11 Kinds of Duck Virus Diseases

Examples

Experimental program
Comparison scheme
Effect test

example 2

Detection for the Specificity of Primer Pairs

I. Preparation of Templates

[0064]1. Extraction of Virus RNA and Acquisition of cDNA

1) Extraction of Virus RNA

[0065]With DNA / RNA Extraction Kit (purchased from TransGen Biotech Co. Ltd., catalog number is ER201), according to kit instructions, RNA of the following virus was extracted respectively (with samples obtained from duck embryo allantoic fluid which were negative for the following virus as negative control samples): avian influenza virus strains: Duck / HK / 717 / 79-d1 (subtype H1N3), Duck / HK / 77 / 76 (subtype H2N3), Duck / HK / 526 / 79 / 2B (subtype H3N6), Duck / HK / 668 / 79 (subtype H4N5), Duck / HK / 531 / 79 (subtype H6N8), Turkey / ont / 6118 / 68 (subtype H8N4), Duck / Guangxi / 1 / 00 (subtype H9N2), Duck / HK / 876 / 80 (subtype H10N3), Duck / HK / 661 / 79 (subtype H11N3), Duck / HK / 862 / 80 (subtype H12N5), Gull / MD / 704 / 77, (subtype H13N5); duck hepatitis virus, duck Tembusu virus, Newcastle disease virus, Muscovy duck reovirus.

2) Acquisition of cDNA

[0066]The RNA samples obt...

example 3

Detection for the Sensitivity of Primer Pairs

I. Preparation of Monoclonal Plasmid Standard and Subtype H7 of Avian Influenza Virus Both Containing Target Gene

[0119]With cDNA or DNA samples of subtype H5 of Avian influenza virus Duck / HK / 313 / 78 (subtype H5N3), subtype H7 of Avian influenza virus Duck / HK / 47 / 76 (subtype H7N2), subtype H9 of Avian influenza virus Duck / Guangxi / 1 / 00 (subtype H9N2), duck hepatitis virus, duck enteritis virus, duck Tembusu virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus as templates, full-sized cDNAs or DNA fragments of gene M of avian influenza virus, gene HA of subtype H5 of avian influenza virus, gene HA of subtype H7 of avian influenza virus, gene HA of subtype H9 of avian influenza virus, gene of region 5′ UTR of duck hepatitis virus, gene UL6 of duck enteritis virus, gene E of duck Tembusu virus, gene L of Newcastle disease virus, gene penton of egg drop syndrome virus, gene SI...

example 4

The Accuracy of Detection with Primer Pairs

[0123]I. The Accuracy of Detection with the Mixture of 12 Primer Pairs

[0124]cDNA samples of subtype H5 of avian influenza virus Duck / HK / 313 / 78 (subtype H5N3) and subtype H7 of avian influenza virus Duck / HK / 47 / 76 (H7N2 subtype) which were both identified by HA gene sequencing were sampled, subtype H9 of avian influenza virus Duck / Guangxi / 1 / 00 (H9N2 type), duck hepatitis virus, duck enteritis virus, duck Tembusu virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus which were identified with common experimental methods, such as: virus isolation, hemagglutination inhibition test, neutralization test etc (cDNA or DNA were extracted with the method used in step I of Example 2) were sampled. cDNA or DNA samples of the above single pathogen, cDNA or DNA samples simulating clinical infection with the above pathogens, mixed template of cDNA or DNA samples of the above pathogens we...

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Abstract

Provided herein is a GeXP detection kit for identification of 11 kinds of duck virus diseases. The detection kit includes a primer set for identifying or auxiliarily identifying pathogens of duck communicable diseases, including one or more of primer pair A, primer pair B primer pair C, primer pair D, primer pair E, primer pair F, primer pair G, primer pair H, primer pair I, primer pair J, primer pair K and primer pair L. The can kit detect, simultaneously avian influenza virus, subtype H5, H7 and H9 of avian influenza virus, duck hepatitis virus, duck enteritis virus, duck Tembusu virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus with the primer set, PCR reagent or primer pairs provided in the present invention.

Description

FIELD OF THE INVENTION[0001]The present invention relates to Biotechnology field, in particular relates to GeXP detection kits for identification of 11 kinds of duck virus diseases.BACKGROUND OF THE INVENTION[0002]Subtypes H5, H7 and H9 of avian influenza virus, duck hepatitis virus, duck enteritis virus, duck Tembusu virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus are 11 kinds of major communicable diseases severely harm to duck. With the development of duck breeding, the incidence of duck communicable virus diseases is also gradually increasing, which has become an important factor which restricts the development of duck breeding. Traditional methods of differential diagnosing these duck communicable diseases mainly include isolation and identification of pathogen, serological tests etc., while these methods are often limited by freshness of clinical samples, pollution degree of clinical samples, or course...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/70
CPCC12Q1/701C12Q2600/158C12Q2600/16C12Q2600/156
Inventor XIE, ZHIXUNZHANG, YANFANGXIE, LIJILIU, JIABOFAN, QINGLUO, SISIDENG, XIANWENXIE, ZHIQINPANG, YAOSHAN
Owner GUANGXI VETERINARY RES INST
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